Supporting Information assembled DNA-encoded ligand ... · 1 Supporting Information Searching for...

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Supporting Information

Searching for avidity by chemical ligation of combinatorially self-assembled DNA-encoded ligand libraries Stefan Matysiakb*, Klaus Hellmuthb, Afaf H. El-Sagheera,c, Arun Shivalingama, Yafuz Ariyurekd, Marco de Jongb, Martine J. Hollestellee, Ruud Outb, Tom Browna*

a Afaf H. El-Sagheer, Arun Shivalingam, Tom Brown Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, Oxford, OX1 3TA, UK.

b Stefan Matysiak, Klaus Hellmuth, Marco de Jong, Ruud Out,

Piculet-Biosciences BV, Galileiweg 8, 2333BD Leiden, the Netherlands.

c Chemistry Branch, Department of Science and Mathematics, Faculty of Petroleum and Mining Engineering, Suez University, Suez 43721, Egypt.

d Yavuz Ariyurek, Leiden Genome Technology Center, Leiden University Medical Center, Leiden, The Netherlands.

e Dr. Martine Hollestelle, Dep. Immunophathology and Blood Coagulation, Sanquin Diagnostic Services, Amsterdam, The Netherlands.

*To whom correspondence should be sent: Tel: +44(0)0865 275413, E-mail: [email protected]

Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2017

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SEQ ID # Name/

Description

Sequence (5' to 3') Spacer Total Length

Other details/ remarks

1 TBA27-T0-c GTCCGTGGTAGGGCAGGTTGGGGTGACAGACTGGTCCGCAAGTTCaaatccaACCTACCTAc T0 62 3'-propargyl dC

2 t-T0-G15D tAGGTAGGTccctataGAACTTGCGGACCAGTCTGGTTGGTGTGGTTGG T0 49 5'-azide-T

3 t-T6-G15D tAGGTAGGTctagtacGAACTTGCGGACCAGTCTTTTTTTGGTTGGTGTGGTTGG T6 55 5'-azide-T

4 t-T12-G15D tAGGTAGGTaccttcgGAACTTGCGGACCAGTCTTTTTTTTTTTTTGGTTGGTGTGGTTGG T12 61 5'-azide-T

5 TBA27s-T0-c GTCCGTCCTACCGCAGCTTCCGTTGACAGACTGGTCCGCAAGTTCaggtctcACCTACCTAc T0 62 3'-propargyl dC

6 t-T12-G15Ds tAGGTAGGTtacacgtGAACTTGCGGACCAGTCTTTTTTTTTTTTTGTGTGTGTGTGTGTG T12 61 5'-azide-T

7 (1 + 2) TBA27-ct-T0-G15DGTCCGTGGTAGGGCAGGTTGGGGTGACAGACTGGTCCGCAAGTTCaaatccaACCTACCTActAGGTAGGTccctataGAACTTGCGGACCAGTCTGGTTGGTGTGGTTGG

T0 111 Clicked

8 (1 + 3) TBA27-ct-T6-G15D

GTCCGTGGTAGGGCAGGTTGGGGTGACAGACTGGTCCGCAAGTTCaaatccaACCTACCTActAGGTAGGTctagtacGAACTTGCGGACCAGTCTTTTTTTGGTTGGTGTGGTTGG

T6 117 Clicked

9 (1 + 4) TBA27-ct-T12-G15D

GTCCGTGGTAGGGCAGGTTGGGGTGACAGACTGGTCCGCAAGTTCaaatccaACCTACCTActAGGTAGGTaccttcgGAACTTGCGGACCAGTCTTTTTTTTTTTTTGGTTGGTGTGGTTGG

T12 123 Clicked

10 (1 + 6) TBA27-ct-T12-G15Ds

GTCCGTGGTAGGGCAGGTTGGGGTGACAGACTGGTCCGCAAGTTCaaatccaACCTACCTActAGGTAGGTtacacgtGAACTTGCGGACCAGTCTTTTTTTTTTTTTGTGTGTGTGTGTGTG

T12 123 Clicked

11 (5 + 2) TBA27s-ct-T0-G15DGTCCGTCCTACCGCAGCTTCCGTTGACAGACTGGTCCGCAAGTTCaggtctcACCTACCTActAGGTAGGTccctataGAACTTGCGGACCAGTCTGGTTGGTGTGGTTGG

T0 111 Clicked

12 (5 + 3) TBA27s-ct-T6-G15DGTCCGTCCTACCGCAGCTTCCGTTGACAGACTGGTCCGCAAGTTCaggtctcACCTACCTActAGGTAGGTctagtacGAACTTGCGGACCAGTCTTTTTTTGGTTGGTGTGGTTGG

T6 117 Clicked

13 (5 + 4) TBA27s-ct-T12-G15D

GTCCGTCCTACCGCAGCTTCCGTTGACAGACTGGTCCGCAAGTTCaggtctcACCTACCTActAGGTAGGTaccttcgGAACTTGCGGACCAGTCTTTTTTTTTTTTTGGTTGGTGTGGTTGG

T12 123 Clicked

14 (5 + 6) TBA27s-ct-T12-G15Ds

GTCCGTCCTACCGCAGCTTCCGTTGACAGACTGGTCCGCAAGTTCaggtctcACCTACCTActAGGTAGGTtacacgtGAACTTGCGGACCAGTCTTTTTTTTTTTTTGTGTGTGTGTGTGTG

T12 123 Clicked

15 G15D GGTTGGTGTGGTTGG NA 15 -

16 TBA27 GTCCGTGGTAGGGCAGGTTGGGGTGAC NA 27 -

17 G15D-T0-TBA27 GGTTGGTGTGGTTGGGTCCGTGGTAGGGCAGGTTGGGGTGAC T0 43directly synthesized

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18 G15D-T6-TBA27 GGTTGGTGTGGTTGGTTTTTTGTCCGTGGTAGGGCAGGTTGGGGTGAC T6 49directly synthesized

19 G15D-T12-TBA27 GGTTGGTGTGGTTGGTTTTTTTTTTTTGTCCGTGGTAGGGCAGGTTGGGGTGAC T12 55directly synthesized

20 TBA27-T0-G15D GTCCGTGGTAGGGCAGGTTGGGGTGACTGGTTGGTGTGGTTGG T0 43directly synthesized

21 TBA27-T6-G15D GTCCGTGGTAGGGCAGGTTGGGGTGACTTTTTTGGTTGGTGTGGTTGG T6 49directly synthesized

22 TBA27-T12-G15DGTCCGTGGTAGGGCAGGTTGGGGTGACTTTTTTTTTTTTGGTTGGTGTGGTTGG T12 55

directly synthesized

23 Reference AGGGATATCACTCAGCATAATGTCGTAC NA 28 Reference

Table S1: Synthesized DNA-Aptamer conjugates: Aptamer sequences are in italics. Underlined regions become double helical after formation of the SABA complexes. Small letter italics indicate the identifier sequence (barcode) of each arm, small letters in a box indicate the position of the triazole linkage or the corresponding terminal nucleotide bearing the reactive groups. Small letter “s” indicates the original aptamer sequence was scrambled. Sequences ID #1-6 are the individual left and right "arms" covalently attached to an aptamer motif or a scrambled reference sequence. Sequences ID #7-14 are the SABA complexes formed after hybridisation and "Click"-reaction, presenting two individual potential binding sequences. Sequence ID #15 and 16 are the monomeric aptamer sequences, which are, together with #23, used as references in the blood clotting assay. Sequences ID #17-22 are directly synthesized, replacing the dsDNA scaffold with an oligo-thymidine linker and were also tested in the blood-clotting assay.

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1. Oligonucleotide Synthesis: DNA sequences SEQ ID #1 and # 5 (Table S1) were synthesized by standard solid-phase

phosphoramidite chemistry using 5'-Dimethoxytrityl-3'-propargyl-5-methyl-2'-deoxycytosine-N-succinyl-

long chain alkylamino-CPG, from GlenResearch. The 5'-azide group for SEQ ID # 2, 3, 4 and 6 were

introduced in a 2-stage process[2]. First 5'-iodo thymidine phosphoramidite was directly added during

solid-phase synthesis. Then the resulting 5'-iodo oligonucleotides were reacted with sodium azide to

complete the transformation. Cleavage of the oligonucleotide from this support requires 2 hr at room

temperature with ammonium hydroxide and complete deprotection requires a further 5 hr at 55oC to

remove the nucleobase protecting groups.

2. Synthesis and Analysis of Self-Assembled Bivalent Aptamers (SABAs):2.i. Synthesis and Purification of SABAs:

CuAAC Reaction ProtocolPrior to chemical ligation, pairs of azide (e.g SEQ ID #2) and alkyne (e.g. SEQ ID #1) oligonucleotides

(100.0 nmol of each) in 0.2 M NaCl (100.0 L) were annealed by heating at 90°C for 5 min and cooling

slowly to room temperature. A solution of CuI catalyst was prepared by adding the tris-

hydroxypropyltriazole ligand[1] (35.0 mol) to sodium ascorbate (50.0 mol in 0.2 M NaCl, 100.0 L)

followed by the addition of CuSO4x5H2O (5.0 mol in 0.2 M NaCl, 50.0 L) under argon. The CuI solution

was added to the annealed oligonucleotide mixture and kept at room temperature for 2 hr under argon.

Reagents were removed by NAP-25 gel-filtration (GE Healthcare) and the ligated product was purified by

anion-exchange HPLC as described previously[2].

The self-assembled bivalent aptamer complexes SEQ ID #7-14 were analysed by 10% PAGE gel

electrophoresis and purified by anion-exchange HPLC on a Gilson HPLC system using a Resource Q

anion-exchange column (6 mL volume, GE Healthcare). The HPLC system was controlled by Gilson

7.12 software, and the following protocol was used: run time, 16 min; flow rate, 5 mL per min; binary

system. Gradient (time in mins (% buffer B)): 0 (0), 3 (0), 4 (40), 9.5 (82), 10 (100), 12 (100), 13 (0), 15.5

(0), 16 (0). Elution buffers: (A) 0.01 M aqueous NaOH, 0.05 M aqueous NaCl, pH 12.0; (B) 0.01 M

aqueous NaOH, 1 M aqueous NaCl, pH 12.0. Elution of oligonucleotides was monitored by ultraviolet

absorption at 295 nm. After HPLC purification oligonucleotides were desalted using a NAP-25 followed

by a NAP-10 Sephadex column (GE Healthcare).

Yields of the purified products after HPLC were 45-57%.

http://www.glenresearch.com/ProductFiles/20-2982.htmlhttp://www.glenresearch.com/ProductFiles/20-2982.htmlhttp://www.glenresearch.com/ProductFiles/10-1931.html

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Figure S1: Denaturing PAGE (10 % polyacrylamide/7 M urea gel) of ligated SABA complexes: Lane 1: SEQ #5 (reference); lane 2: CuAAC reaction mixture (unpurified) to yield SEQ ID #11; lane 3: SEQ ID #1 (reference); lane 4: CuAAC reaction mixture (unpurified) to yield SEQ ID #7. Constant power of 20 W using 0.09 M Tris-borate-EDTA buffer (pH 8.0).

SEQ ID # Calc. Mass Found. Mass

1 19212 19211

5 18930 18929

2 15271 15270

3 17136 17135

4 18937 18938

6 18936 18936

7 34482 34482

8 36348 36347

9 38149 38151

10 38148 38148

11 34201 34201

12 36066 36066

13 37867 37866

14 37866 37868

Table S2. Mass spectrometry analysis of the monovalent precursors SEQ ID #1-6 and the corresponding click-ligated products SEQ ID #7-14. Mass spectra were recorded on a Bruker micrOTOFTM II focus ESI-TOF MS instrument in ES- mode and fit well with the calculated values.

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2.ii. Gel-electrophoretic Analysis of Self-Assembled Bivalent Aptamers SEQ ID #7-14

L 7 8 9 10 11 12 13 14

Figure S2: Gel-electrophoretic analysis of self-assembled bivalent aptamers: The self-assembled bivalent aptamers SEQ ID #7-14 were dissolved in TE buffer (100 mM Tris pH 8.0, 1 mM EDTA). Their concentration was determined via UV absorption at 260 nm (NanoDrop, Thermo Scientific), then diluted to 10 ng/µl. 1 µl of each single-stranded DNA was loaded into an Agilent smallRNA chip and run on the Agilent BioAnalyzer 2100 capillary electrophoresis system (L= small RNA ladder). The self-assembled bivalent aptamers run faster than expected size. This is an indication for the stability of the self complementary assembly-region even under denaturing buffer conditions, which are sufficient to dissolve most of the secondary structures of RNA molecules(http://www.chem.agilent.com/Library/usermanuals/Public/G2938-90094revB_QG_SmallRNA.pdf).

http://www.chem.agilent.com/Library/usermanuals/Public/G2938-90094revB_QG_SmallRNA.pdf

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2.iii. PCR amplification and Sanger Sequencing of individual self-assembled bivalent aptamers:

PCR amplification Scheme for SEQ ID #8:

Figure S4: Example of a "Click"- reaction followed by PCR amplification: SEQ ID #1 (left arm) is covalently conjugated with SEQ ID #3 (right arm) as described to yield SEQ ID #8, which folds into the corresponding self-assembled bivalent aptamer. Clicked termini in small litalics boxed. The corresponding amplification via PCR using only one universal primer sequence ID # 24 yields SEQ ID #27 (sense strand) and SEQ ID #28 (antisense strand). The self-complementary parts of the strands are underlined. Aptamer motif and spacer specific sequences (barcode) in small italics. Clicked site now indicated in bold capital italics.

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Analysis of PCR products SEQ IDs # 25-40

SEQ ID #

Name Sequence (5' to 3')

24 U18_primer AGACTGGTCCGCAAGTTC

25 PCR product(forward)from template SEQ ID #7

AGACTGGTCCGCAAGTTCaaatccaACCTACCTACTAGGTAGGTccctataGAACTTGCGGACCAGTCT

26 PCR product(reverse complement)from template SEQ ID #7

AGACTGGTCCGCAAGTTCtatagggACCTACCTAGTAGGTAGGTtggatttGAACTTGCGGACCAGTCT

27 PCR product(forward)from SEQ ID #8

AGACTGGTCCGCAAGTTCaaatccaACCTACCTACTAGGTAGGTctagtacGAACTTGCGGACCAGTCT

28 PCR product(reverse complement)from SEQ ID #8

AGACTGGTCCGCAAGTTCgtactagACCTACCTAGTAGGTAGGTtggatttGAACTTGCGGACCAGTCT


AGACTGGTCCGCAAGTTCaaatccaACCTACCTACTAGGTAGGTaccttcgGAACTTGCGGACCAGTCT


AGACTGGTCCGCAAGTTCcgaaggtACCTACCTAGTAGGTAGGTtggatttGAACTTGCGGACCAGTCT


AGACTGGTCCGCAAGTTCaaatccaACCTACCTACTAGGTAGGTtacacgtGAACTTGCGGACCAGTCT

32 PCR product(reverse complement)from SEQ ID #10)

AGACTGGTCCGCAAGTTCacgtgtaACCTACCTAGTAGGTAGGTtggatttGAACTTGCGGACCAGTCT


AGACTGGTCCGCAAGTTCaggtctcACCTACCTACTAGGTAGGTccctataGAACTTGCGGACCAGTCT


AGACTGGTCCGCAAGTTCtatagggACCTACCTAGTAGGTAGGTgagacctGAACTTGCGGACCAGTCT

35 PCR product(Fwd)from SEQ ID #12

AGACTGGTCCGCAAGTTCaggtctcACCTACCTACTAGGTAGGTctagtacGAACTTGCGGACCAGTCT


AGACTGGTCCGCAAGTTCgtactagACCTACCTAGTAGGTAGGTgagacctGAACTTGCGGACCAGTCT


AGACTGGTCCGCAAGTTCaggtctcACCTACCTACTAGGTAGGTaccttcgGAACTTGCGGACCAGTCT


AGACTGGTCCGCAAGTTCcgaaggtACCTACCTAGTAGGTAGGTgagacctGAACTTGCGGACCAGTCT


AGACTGGTCCGCAAGTTCaggtctcACCTACCTACTAGGTAGGTtacacgtGAACTTGCGGACCAGTCT


AGACTGGTCCGCAAGTTCacgtgtaACCTACCTAGTAGGTAGGTgagacctGAACTTGCGGACCAGTCT

Table S4: List of PCR products SEQ IDs #25-40 generated from templates SEQ IDs #7-14All sequences listed in the 5' to 3' orientation. Self-complementary part of the strand underlined. Sequence barcodes in italics. “Clicked” termini in the sense as well as reverse complementary site in the antisense strand are annotated in bold capital italics.

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ladder 25&26 27&28 29&30 31&32 33&34 35&36 37&38 39&40 blank

Figure S5: Gel-electrophoretic analysis of PCR products SEQ ID # 25-40 from SEQ ID #7-14. Only one primer was used (sequence U18 (SEQ ID #24)).10 ng template, 1.2 μM primer, 0.5 mM triphosphates and 2.5 U thermostable DNA polymerase were mixed and PCR amplification was executed with 24 cycles at 600C annealing temperature. Gel-electrophoretic analysis was done on a Bioanalyzer 2100 DNA -1000 chip with 1 μl PCR product for each lane.

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Sanger Sequencing of PCR products

PCR product I



PCR II

GTCACTCCTGGTCCGCAAGTTCgta

SEQ ID# 43 Y7b_U15_S3AGCTGTG

CTGGTCCGCAAGTTCaaa

SEQ ID #41 Y7a_U15_S3

AGCTGTGCTGGTCCGCAAGTTCaaatccaACCTACCTACTAGGTAGGTctagtacGAACTTGCGGACCAGGAGTGAC

GTCACTCCTGGTCCGCAAGTTCgtactagACCTACCTAGTAGGTAGGTtggatttGAACTTGCGGACCAGCACAGCT

PCR product II

PCR III

SEQ ID #63 InPERead1_Y7a_U15



ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGCTGTGCTGGTCCGCAAGTTC

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTCACTCCTGGTCCGCAAGTTC

SEQ ID# 64 InPERead2_Y7b_U15

PCR product III

ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGCTGTGCTGGTCCGCAAGTTCaaatccaACCTACCTACTAGGTAGGTctagtacGAACTTGCGGACCAGGAGTGACAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTCACTCCTGGTCCGCAAGTTCgtactagACCTACCTAGTAGGTAGGTtggatttGAACTTGCGGACCAGCACAGCTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT

SEQ ID# 49 SEQ ID# 50

SEQ ID #66

SEQ ID #65

SEQ ID# 27 (PCR Product I, fwd)

SEQ ID# 28 (PCR Product I, rev. compl.)

PCR IV

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT.....AGATCGGAAGAGCACACGTCTGAACTCCAGTCACatcacgATCTCGTATGCCGTCTTCTGCTTG

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT.....AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT

ACACTCTTTCCCTACACGACGCTCTTCCGATCT.....AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC

SEQ ID #67 InPE1.0

CAAGCAGAAGACGGCATACGAGATcgtgatGTGACTGGAGTTC

SEQ ID #68 InPEIndex_1

CAAGCAGAAGACGGCATACGAGATcgtgatGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT.....AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT

SEQ ID #71

SEQ ID #72

PCR product III

SEQ ID #65

SEQ ID #66

PCR product IV

PCR Product IV

AATGATACGGCGACCACC

SEQ ID # 73 SangerSeqPrimer_Read1

CAAGCAGAAGACGGCATA

SEQ ID # 74 SangerSeqPrimer_Read2


CAAGCAGAAGACGGCATACGAGATcgtgatGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT.....AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT

Sanger Seq Data Analysis

aatcca ctagtac atcacg

BC1 BC3 Idx1

SEQ ID #71

SEQ ID #72

Figure S6: Sample Preparation prior to Sanger Sequencing. As an example for sample preparation for Sanger sequencing the scheme for PCR product SEQ ID #27 and #28 is shown. PCR products SEQ ID #25, 26 and #29-40 are processed accordingly. Preparation of SEQ ID #71 and #72, which are sequenced using sequencing primers SEQ ID #73 and #74 is shown. Sequencing was done on a 96-capillary 3730xl DNA Analyzer (Life Technologies). The individual samples were processed using the corresponding BigDye® Direct Cycle Sequencing Kit and protocol.

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41 Y7a_U15_BC1f AGCTGTGCTGGTCCGCAAGTTCaaa

42 Y7b_U15_BC2r GTCACTCCTGGTCCGCAAGTTCtat

43 Y7b_U15_BC3r GTCACTCCTGGTCCGCAAGTTCgta

44 Y7b_U15_BC4r GTCACTCCTGGTCCGCAAGTTCcga

45 Y7a_U15_BC5f AGCTGTGCTGGTCCGCAAGTTCagg

46 Y7b_U15_BC6r GTCACTCCTGGTCCGCAAGTTCacg

47 PCR product II(forward)of PCR product I of SEQ ID#7 (SEQ ID #25 and #26) using primer SEQ ID #41

AGCTGTGCTGGTCCGCAAGTTCaaatccaACCTACCTACTAGGTAGGTccctataGAACTTGCG

GACCAGGAGTGAC

48 PCR product II(reverse-complement) of PCR product I of SEQ ID #7 (SEQ ID #25 and #26.using primer SEQ ID #42

GTCACTCCTGGTCCGCAAGTTCtatagggACCTACCTAGTAGGTAGGTtgcatttGAACTTGCGGACCAGCACAGCT

49 PCR product II(forward)of PCR product I of SEQ ID#8 (SEQ ID #27 and #28) using primer SEQ ID #41.


50 PCR product II(reverse-complement) of PCR product I of SEQ ID #8 (SEQ ID #27 and #28)using primer SEQ ID #43.


51 PCR product II(forward)of PCR product I of SEQ ID#9 (SEQ ID #29 and #30) using primer SEQ ID #41.

AGCTGTGCTGGTCCGCAAGTTCaaatccaACCTACCTACTAGGTAGGTaccttcgGAACTTGCGGACCAGGAGTGAC

52 PCR product II(reverse-complement)of PCR product I of SEQ ID #9 (SEQ ID #29 and #30)using primer SEQ ID #44.

GTCACTCCTGGTCCGCAAGTTCcgaaggtACCTACCTAGTAGGTAGGTtggatttGAACTTGCGGACCAGCACAGCT

53 PCR product II(forward) of PCR product I of SEQ ID#10 (SEQ ID #31 and #32) usingprimer SEQ ID #41.

AGCTGTGCTGGTCCGCAAGTTCaaatccaACCTACCTACTAGGTAGGTtacacgtGAACTTGCGGACCAGGAGTGAC

54 PCR product II(reverse-complement) of PCR product I of SEQ ID#10 (SEQ ID #31 and #32)using primer SEQ ID #46.

GTCACTCCTGGTCCGCAAGTTCacgtgtaACCTACCTAGTAGGTAGGTtggatttGAACTTGCGGACCAGCACAGCT

55 PCR product II(forward) of PCR product I of SEQ ID#11 (SEQ ID #33 and #34) using primer SEQ ID #45.

AGCTGTGCTGGTCCGCAAGTTCaggtctcACCTACCTACTAGGTAGGTccctataGAACTTGCGGACCAGGAGTGAC


GTCACTCCTGGTCCGCAAGTTCtatagggACCTACCTAGTAGGTAGGTgagacctGAACTTGCGGACCAGCACAGCT


AGCTGTGCTGGTCCGCAAGTTCaggtctcACCTACCTACTAGGTAGGTctagtacGAACTTGCGGACCAGGAGTGAC


GTCACTCCTGGTCCGCAAGTTCgtactagACCTACCTAGTAGGTAGGTgagacctGAACTTGCGGACCAGCACAGCT


AGCTGTGCTGGTCCGCAAGTTCaggtctcACCTACCTACTAGGTAGGTaccttcgGAACTTGCGGACCAGGAGTGAC


GTCACTCCTGGTCCGCAAGTTCcgaaggtACCTACCTAGTAGGTAGGTgagacctGAACTTGCGGACCAGCACAGCT


AGCTGTGCTGGTCCGCAAGTTCaggtctcACCTACCTACTAGGTAGGTtacacgtGAACTTGCGGACCAGGAGTGAC

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GTCACTCCTGGTCCGCAAGTTCacgtgtaACCTACCTAGTAGGTAGGTgagacctGAACTTGCGGACCAGCACAGCT

63 InPEREad1_Y7a_U15 ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGCTGTGCTGGTCCGCAAGTTC

64 InPEREAd2_Y7b_U15 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTCACTCCTGGTCCGCAAGTTC

65 Pre-multiplexed PCR III product (forward) ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGCTGTGCTGGTCCGCAAGTTCAAATCCAACCTACCTACTAGGTAGGTCTAGTACGAACTTGCGGACCAGGAGTGACAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC

66 Pre-multiplexed PCR III product (anti-sense) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTCACTCCTGGTCCGCAAGTTCGTACTAGACCTACCTAGTAGGTAGGTTGGATTTGAACTTGCGGACCAGCACAGCTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT

67 InPE1.0 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT

68 InPEIndex_1 multiplex/index1 CAAGCAGAAGACGGCATACGAGATcgtgatGTGACTGGAGTTC

69 InPEIndex_2 multiplex/index2 CAAGCAGAAGACGGCATACGAGATacatcgGTGACTGGAGTTC

70 InPEIndex_3 multiplex/index3 CAAGCAGAAGACGGCATACGAGATgcctaaGTGACTGGAGTTC

71 PCR product IV(forward) AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT.....AGATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG

72 PCR product IV (reverse-complement) CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT.....AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT

73 Sanger Sequencing Primer InPE1-18 AATGATACGGCGACCACC

74 Sanger Sequencing Primer InPE2-18 CAAGCAGAAGACGGCATA

Table S5: Primers and corresponding PCR products used for Sanger Sequencing.All sequences in 5' to 3' orientation. Small italics indicate the individual identifier sequence (barcode), underlined letters indicate reverse complementary sequences. Not all PCR products of PCR III and IV are listed, except #65, 66 (III) and #71, 72 (IV) used as examples for the general scheme above.

Figure S7: Results of Sanger-sequencing with Primer SEQ ID #73 and #74 of PCR products SEQ ID #71 and 72. The corresponding identifier sequences (SI1 = aaatcca) and (SI3= ctagtac) can be read-out correctly on both strands. Indexing sequence (atcacg), which is introduced to allow next generation sequencing on an Illumina MiSeq Platform in a separate experiment after multiplexing, can be read-out correctly on forward strand (not shown).

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3. Biological Activity

3.i. Blood Clotting ExperimentsSamples were obtained with consent from Sanquin Bloodbank. Pooled plasma from a pool of more than

32 healthy adult donors was used.

To evaluate the inhibitory potency of individual nucleic acid ligand or bivalent complex thereof, we

measured the clotting time of each sample containing only thrombin, the ligand or ligand complex and

fibrinogen substrate in physiological buffer. The mixture of sample becomes non fluidic when the

fibrinogen is digested by thrombin. As a result, the different timepoints of this transition can be used as

an indicator. Briefly, 1μl of 10 μM thrombin and 1 μl of 100 μM monovalent or bivalent aptamer were

added to a disposable transparent plastic cuvette (Fisher Scientific) containing 200 μl buffer and

incubated for 15 min. Then, 4 μl of 20 mg/ml fibrinogen was added, and samples in the cuvette were

carefully examined by tilting the cuvette to record the time when the sample became non fluidic. Each

experiment was performed in tandem. A reaction mixture containing only thrombin and fibrinogen was

always tested together with other samples as an internal standard. All clotting times were normalized

based on the internal standard and compared with it.

To evaluate the feasibility of the self-assembled bivalent aptamer as a potential anticoagulant reagent,

Standard activated Partial Thromboplastin Time (aPTT)[3] and Prothrombin Time (PT)[4] values for each

individual or ligand composition were determined by using human plasma samples. Procedures applied

were those recommended by the supplier. For aPTT determination, 50 μl UCRP was preincubated at

37°C with a different amount of each ligand for 2 min; then 50 μl aPPT-L was added and incubated for

another 200 sec. Next, 50 μl of pre-warmed CaCl2 was added to initiate the intrinsic clotting cascade.

Finally, the scattering signal was monitored until the signal was saturated. For PT determination, 50 μl of

UCRP was pre-incubated at 37°C with a different amount of each ligand or ligand complex for 2 min;

then 50 μl of thromboplastin-L was added to initiate the extrinsic clotting cascade. Finally, the scattering

signal was monitored until the signal was saturated. For the calculation of aPTT and PT, the end time

was determined to be the point where scattering signal reached half maximum between lowest and

maximum points. This was repeated twice, and each set of experiments was done with one batch of

plasma.

14

PBS 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 2325

35

45

55

9

10

11

12

13aPTT PTAP

TT (s

ec)

PT (s

ec)

Figure S8: Standard Prothrombin Time (PT) and activated Partial Thromboplastin time (aPTT) values for each individual or aptamer ligand composition: The values were determined by using human plasma samples. Whilst sequences do not show any significant difference in the PT values, except #15, the benefit of combining two binding motifs is clearly evident in the aPTT values. The aPTT value of a non-Thrombin binding oligonucleotide sequence SEQ ID # 23 is fully identical to the reference (buffer) below 30 sec. Combinations of SEQ ID #15 and #16 lead to values above 35 sec depending on the distance between the two aptamers. SEQ ID #17-19 and #20-22 are covalently conjugated aptamer motifs #15 and #16 without and with a T6 or T12 spacer. Similar aPTT values can be seen as confirmation of a stable dsDNA stem for the self-assembled bivalent ligand complexes (SABAs) of two binding motifs with varied distances (SEQ ID #7,8 and 9). Further more mutation of one of the binding motifs lead to decreased aPTT values as expected (SEQ ID #10-14).

ID # Sequence Description PT aPTT7 TBA27-ct-T0-G15D 10.7 35.38 TBA27-ct-T6-G15D 11.6 52.69 TBA27-ct-T12-G15D 11.2 51.110 TBA27-ct-T12-G15Ds 10.8 43.011 TBA27s-ct-T0-G15D 10.9 34.712 TBA27s-ct-T6-G15D 10.9 36.713 TBA27s-ct-T12-G15D 11.2 43.614 TBA27s-ct-T12-G15Ds 10.5 33.815 G15D 12.6 36.016 TBA27 10.8 35.017 G15D-T0-TBA27 11.7 42.318 G15D-T6-TBA27 12.2 48.919 G15D-T12-TBA27 12.6 52.820 TBA27-T0-G15D 11.4 42.621 TBA27-T6-G15D 11.6 45.722 TBA27-T12-G15D 12.0 51.623 Reference 10.6 29.1

Table S6. Sequences used for blood clotting experiments and the corresponding PT and aPTT values (average from at least two technical replicates, 2-4 data points in total).

15

4. Aptamer selection, amplification and sequencing

4.i. Self-assembled bivalent aptamer (SABA) model library creationA model library was prepared using SABA sequences ID #7-14. SEQ ID # 7-13 were mixed in 1:1 ratio

and background DNA SEQ ID #14 (200 pmol) was added. The final mix comprised 99.125% background

DNA and 0.125 % of each of the SEQ ID #7-13. 1 M was the final concentration.

4.ii Protein ImmobilisationMagnetic Dynabeads® MyOne Carboxylic Acid (Invitrogen, cat. N0 65011) are resuspended by rolling

the vial for 30 min on a rotor at 20 rpm, and 0.5 ml suspension was transferred to a new tube. The tube

was placed close to a magnet for 1 min, the supernatant was removed and the beads were washed two

times with 0.5 ml MEST buffer (50 mM 2-(N-morpholino)ethanesulfonic acid, 0.01 % Tween 20, pH 6.0).

The beads were resuspended in 50 μl MEST buffer and activated by addition of 50 μl EDC (10 mg/ml N-

(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride, Sigma E6383) for 30 min at room

temperature on a rotor at 10 rpm. The supernatant was removed, the beads were resuspended in 100 μl

MEST buffer and split into two 50 μl aliquots. The first aliquot was incubated with 6 nmol corresponding

to 200 μg -thrombin (Tb, Haematologic Technologies, HCT-0020) in 250 μl MEST buffer (20 µM

thrombin final), the second aliquot was blocked with 5 mM aminoethanol (EA) in 250 μl MEST buffer for

2.5 h on a rotor at 10 rpm at room temperature. The tubes were placed close to a magnet for 1 min, the

supernatants were removed and the beads were each washed two times with 0.25 ml PBSMT buffer

(137 mM sodium chloride, 2.7 mM potassium chloride and 10 mM sodium/potassium phosphate buffer

solution pH7.4, Ambion AM9624, supplemented with 1 mM magnesium chloride and 0.01 % Tween 20).

4.iii. Selection by Bead CaptureThrombin-coupled (Tb) beads and negative control (EA = deactivated with ethanolamine) beads were

each re-suspended in 160 μl PBSMT, and to each aliquot 40 μl of 5 μM (corresponding to 200 pmol)

SABA library was added (1 μM SABA library final). The tubes containing the mixtures were incubated for

1 h on a rotor at 10 rpm at room temperature. The tubes were placed close to a magnet for 1 min, the

supernatants were removed and the beads were washed two times with 0.25 ml PBSMT buffer. The

beads were boiled each in 100 μl of 1x thermostable DNA buffer (Roboklon) for 5 min at 95 oC in a

thermocycler and cooled down to room temperature. The tubes were placed close to a magnet for 1 min,

and 90 μl of the supernatants were transferred to new tubes for further analysis.

4.iv. Universal PCR Amplification The bead-selected (Tb and EA) members of the SABA library were universally amplified by the following

semi-quantitative PCR protocol:

16

20 µl of DNAse/RNAse free water (Life Technologies) was supplemented with 3 µl of 10x buffer for

thermostable DNA polymerase (Roboklon), 6 µl of 10 µM primer U18 (SEQ ID # 24; Biolegio), 0.5 µl of

25 mM dNTP mix (25 mM of each deoxyribonucleotide dATP, dCTP, dGTP, dTTP; Invitrogen), 0.5 µl of

thermostable DNA polymerase (5 U/µl; Roboklon) and 20 µl of template DNA in 1x thermostable DNA

buffer (or 1 x thermostable DNA buffer alone for blank analysis). Template DNA for the standard curve

was a serial dilution of the SABA library (0.5 µM to 0.5 pM) in 1x thermostable DNA buffer, template DNA

for selection analysis consisted of bead eluate in 1x thermostable DNA buffer. The resulting 50 µl PCR

premixes were incubated in a thermocycler instrument (BioRad iCycler) with the following program

settings: 1x melt 03:00 min 95 ºC, 24x melt 00:30 min 95 ºC, 24x anneal 00:30 min 60 ºC, 24x elongate

00:30 min 72 ºC 1x elongate 03:00 min 72 ºC, 1x hold 4 ºC. 1 µl of the PCR mix was analyzed by

capillary electrophoresis in a Bioanalyzer DNA-1000 Lab-on-a-Chip (Agilent). The concentration of the

PCR products were determined by peak integration, performed by the Bioanalyzer 2100 Expert software,

version B.02.08.SI648 (Agilent).

L 1000 100 10 0 fmol Tb EA Tb EA beads2x 2x 6x 6x wash

pre-Capture post-Capture 24 PCR cycles 36 PCR cycles

Figure S9: Semi-quantitative 1st PCR with Single Primer U18: According to the Agilent BioAnalzyer Data (described above) the PCR detection limit for SABA hairpins with single universal U18 primer is 10 fmol. Apparently selection of a SABA library on DynaBeads coupled to 20 µg thrombin results in captured material in the low fmol range (2x wash) and much lower range (6x wash). Negative selection on beads blocked with ethanolamine results in very little (2x wash) and nearly non detectable material (6x wash).

4.v. Quantitative PCR (qPCR) AnalysisThe compositions of the universal PCR products of the bead-selected SABAs (Tb and EA) were

determined by amplification of individual SABA barcodes by the following quantitative PCR (qPCR)

protocol: 6 µl of DNAse/RNAse free water (Life Technologies) were supplemented with 10 µl of iQ™

SYBR®Green Supermix (Bio-Rad, cat. # 170-8882), 1 µl of 10 µM forward primer (SEQ ID # 75, 79 and

81; Biolegio), 1 µl of 10 µM reverse primer (SEQ ID #76-78, 80, 82; Biolegio) and 2 µl of 60 nM

(corresponding to 120 fmol) template DNA. The resulting 20 µl PCR premixes were incubated in a

thermocycler instrument (Bio-Rad iCycler) with the following program settings: 1x melt 03:00 min 95 ºC,

40x melt 00:30 min 95 ºC, 40x anneal 00:30 min 60 ºC, 40x elongate 00:30 min 72 ºC. The cycle

threshold (Ct) values were calculated by PCR baseline subtraction curve fits of the SYBR®Green

amplification charts, performed by the iQ™5 Optical System Software, Version 2.1 (Bio-Rad).

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Enrichment factors (Ef) were calculated in Microsoft Excel as follows: Ef=POWER(1,7;Ct(EA-

Tb))=POWER(1,7;AVERAGE(Ct(EA))-AVERAGE(Ct(Tb))).

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SEQ ID # Name Sequence (5΄ to 3´)

24 U18_primer AGACTGGTCCGCAAGTTC

75 U11_S7-BC1f barcode 1 specific TCCGCAAGTTCaaatcca

76 U11_S7-BC2r barcode 2 specific TCCGCAAGTTCtataggg

77 U11_S7-BC3r barcode 3 specific TCCGCAAGTTCgtactag

78 U11_S7-BC4r barcode 4 specific TCCGCAAGTTCcgaaggt

79 U11_S7-BC5f barcode 5 specific TCCGCAAGTTCaggtctc

80 U11_S7-BC6r barcode 6 specific TCCGCAAGTTCacgtgta

81 Y7a_U15_U3f background specific AGCTGTGCTGGTCCGCAAGTTCctg

82 Y7b_U15_U3r background specific GTCACTCCTGGTCCGCAAGTTCgtc

83 PCR product II(forward)from SEQ ID #25 and 26 and primer #75

TCCGCAAGTTCaaatccaACCTACCTACTAGGTAGGTccctataGAACTTGCGGA

84 PCR product II(reverse complement) from SEQ ID #25 and 26and primer #76

TCCGCAAGTTCtatagggACCTACCTAGTAGGTAGGTtgcatttGAACTTGCGGA

85PCR product II(forward) from SEQ ID #27 and 28and primer #75 TCCGCAAGTTCaaatccaACCTACCTACTAGGTAGGTctagtacGAACTTGCGGA

86PCR product(reverse complement)from SEQ ID #27 and 28and primer #77 TCCGCAAGTTCgtactagACCTACCTAGTAGGTAGGTtggatttGAACTTGCGGA

87PCR product (forward)from SEQ ID #29 and 30and primer #75 TCCGCAAGTTCgtactagACCTACCTAGTAGGTAGGTtggatttGAACTTGCGGA

88PCR product (reverse complement)from SEQ ID #29 and 30and primer #78

TCCGCAAGTTCcgaaggtACCTACCTAGTAGGTAGGTtggatttGAACTTGCGGA

89PCR product (forward)from SEQ ID #31 and 32and primer #75 TCCGCAAGTTCaaatccaACCTACCTACTAGGTAGGTtacacgtGAACTTGCGGA


TCCGCAAGTTCacgtgtaACCTACCTAGTAGGTAGGTtggatttGAACTTGCGGA

91PCR product (forward)from SEQ ID #33 and 34and primer #79 TCCGCAAGTTCaggtctcACCTACCTACTAGGTAGGTccctataGAACTTGCGGA


TCCGCAAGTTCtatagggACCTACCTAGTAGGTAGGTgagacctGAACTTGCGGA

93PCR product (forward)from SEQ ID #35 and 36and primer #79 TCCGCAAGTTCaggtctcACCTACCTACTAGGTAGGTctagtacGAACTTGCGGA


TCCGCAAGTTCgtactagACCTACCTAGTAGGTAGGTgagacctGAACTTGCGGA

95PCR product (forward)from SEQ ID #37 and 38and primer #79 TCCGCAAGTTCaggtctcACCTACCTACTAGGTAGGTaccttcgGAACTTGCGGA


TCCGCAAGTTCcgaaggtACCTACCTAGTAGGTAGGTgagacctGAACTTGCGGA

97PCR product (forward) from SEQ ID #39 and 40and primer #81 AGCTGTGTCCGCAAGTTCaggtctcACCTACCTACTAGGTAGGTtacacgtGAACTTGCGGAC


GTCACTGTCCGCAAGTTCacgtgtaACCTACCTAGTAGGTAGGTgagacctGAACTTGCGGACCAGTCT

Table S7: List of primer sequences used for qPCR (SEQ ID #24, 75-82) and corresponding PCR products (forward and reverse complement). All sequences in 5' to 3' orientation. Small italics indicate the individual identifier sequence (barcode), underlined letters indicate reverse complementary sequences.

19



qPCR

TCCGCAAGTTCgtactag

SEQ ID # 77 U11_S7-BC3rTCCGCAAGTTCaaatcca

SEQ ID #75 U11_S7-BC1f

TCCGCAAGTTCaaatccaACCTACCTACTAGGTAGGTctagtacGAACTTGCGGA

TCCGCAAGTTCgtactagACCTACCTAGTAGGTAGGTtggatttGAACTTGCGGA

PCR product I mix

GTCCGTGGTAGGGCAGGTTGGGGTGACAGACTGGTCCGCAAGTTCaaatccaACCTACCTActAGGTAGGTctagtacGAACTTGCGGACCAGTCTTTTTTTGGTTGGTGTGGTTGG

AGACTGGTCCGCAAGTTC

SEQ ID # 24 (U18 primer)

Template SEQ ID #8 (from SEQ ID #7 to 14 mix)

PCR I

qPCR products SEQ ID #85 and #86

Template SEQ ID #27

Template SEQ ID #28

Figure S10: Sample Preparation for quantitative PCR was done according to the above scheme and protocol. As an example the scheme is shown in detail starting with template SEQ ID #8 from the mix SEQ ID#7-14.

Results:

Figure S11: qPCR Data: Ct (Tb-EA) values of the different SABAs show enrichment for SEQ ID # 7-9 as well as for #12 and #13. From this data it can be concluded that SEQ ID #8 with a T6 spacer and #9 with a T12 spacer is most efficient combination of binding motifs. If only one binding motif is scrambled the numbers of the corresponding SABA after selection are less. Modification (scrambled sequence) of TBA27 (#12,13) is less effective than modification of G15D (#10 and #11). The relative abundance of SEQ ID #14, where both sequences have been modified, is dramatically reduced.

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4.vi. Illumina Multiplexing PCR

The universally amplified members of the SABA library (Tb and EA) were further amplified by the

following PCR protocol: 36 µl of DNAse/RNAse free water (Life Technlogies) was supplemented with 5

µl of 10x thermostable DNA Buffer (Roboklon), 3 µl of each 10 µM primer (Biolegio), 0.5 µl of 25 mM

dNTP mix (25 mM of each deoxyribonucleotide dATP, dCTP, dGTP, dTTP; Invitrogen), 0.5 µl of

thermostable DNA polymerase (5 U/µl; Roboklon) and 2 µl of template DNA (or 2 µl PCR buffer alone for

blank analysis). The resulting 50 µl PCR premixes were incubated in a thermocycler instrument (BioRad

iCycler) with the following program settings: 1x melt 03:00 min 95 ºC, 18x melt 00:30 min 95 ºC, 18x

anneal 00:30 min 65 ºC, 18x elongate 00:30 min 72 ºC 1x elongate 03:00 min 72 ºC, 1x hold 4 ºC. 1 µl of

the PCR mix was analyzed by capillary electrophoresis in a Bioanalyzer DNA-1000 Lab-on-a-Chip

(Agilent). The concentration of the PCR products were determined by peak integration, performed by the

Bioanalyzer 2100 Expert software, version B.02.08.SI648 (Agilent).

SEQ ID Name Sequence (5' to 3')

68 InPEIndex_1 multiplex/index1 CAAGCAGAAGACGGCATACGAGATcgtgatGTGACTGGAGTTC

69 InPEIndex_2 multiplex/index2 CAAGCAGAAGACGGCATACGAGATacatcgGTGACTGGAGTTC

70 InPEIndex_3 multiplex/index3 CAAGCAGAAGACGGCATACGAGATgcctaaGTGACTGGAGTTC

99 IllumSeqPrimer_Read1 ACACTCTTTCCCTACACGACGCTCTTCCGATCT

100 IllumSeqPrimer_Read2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT

101 IllumSeqPrimer_IndexRead GATCGGAAGAGCACACGTCTGAACTCCAGTCAC

Table S8: Primers used for NGS sample preparation after selection and multiplexing. Other sequences mentioned in the scheme (Figure S12) are listed previously in Table S5. All sequences in 5' to 3' orientation. Small italics indicate the individual identifier sequence (index).

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Illumina Sequencing:

PCR product I (SEQ IDs #27&28) of SABA 8 (SEQ ID #8)



PCR II (first pre-multiplexing)

GTCACTCCTGGTCCGCAAGTTCgta

SEQ ID# 43 Y7b_U15_S3AGCTGTG

CTGGTCCGCAAGTTCaaa

SEQ ID #41 Y7a_U15_S3



PCR product II

PCR III (second pre-multiplexing)

SEQ ID #63 InPERead1_Y7a_U15



ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGCTGTGCTGGTCCGCAAGTTC

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTCACTCCTGGTCCGCAAGTTC

SEQ ID# 64 InPERead2_Y7b_U15

PCR product III

ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGCTGTGCTGGTCCGCAAGTTCaaatccaACCTACCTACTAGGTAGGTctagtacGAACTTGCGGACCAGGAGTGACAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTCACTCCTGGTCCGCAAGTTCgtactagACCTACCTAGTAGGTAGGTtggatttGAACTTGCGGACCAGCACAGCTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT

SEQ ID# 49 SEQ ID# 50

SEQ ID #66

SEQ ID #65

SEQ ID# 27 (PCR Product I, fwd)

SEQ ID# 28 (PCR Product I, rev.compl.)

Multiplexing

PCR IV (multiplexing)


Multiplexed PCR Product IV (not all listed, except SEQ ID #71 and #72)

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT.....AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT

ACACTCTTTCCCTACACGACGCTCTTCCGATCT.....AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC

SEQ ID #67 InPE1.0

CAAGCAGAAGACGGCATACGAGATcgtgatGTGACTGGAGTTC

SEQ ID #68 InPEIndex_1CAAGCAGAAGACGGCATACGAGATcgtgatGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT.....AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT

PCR product III

SEQ ID #71

SEQ ID #72

ACACTCTTTCCCTACACGACGCTCTTCCGATCTSEQ ID #99 IllumSeqPrimer_Read1

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT

GATCGGAAGAGCACACGTCTGAACTCCAGTCAC

SEQ ID #101 IllumSeqPrimer_IndexRead

NGS Data Analysis

SEQ ID100 xx IllumSeqPrimer_Read2

CAAGCAGAAGACGGCATACGAGATcgtgatGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC......AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT

aatcca ctagtac atcacg

BC1 BC3 Idx1

SEQ ID #71

SEQ ID #72


Multiplexed PCR Products IV (not all listed, except SEQ ID #71 and #72)

Figure S12:

22

As an example for sample preparation for Illumina NGS sequencing the scheme for PCR amplification is of SABA oligonucleotide #8 is shown. After selection a first PCR amplification with universal primer U18 (SEQ ID #24) is performed to yield PCR product I (SEQ IDs #27 and #28, see Figure S10.) For a second PCR amplification eight aliquots of PCR product I were amplified with eight primer pairs (forward and reverse,e.g. SEQ IDs #41 and #43) to yield PCR product II (SEQ IDs #49 and #50). This is followed by a third PCR amplification with universal primer pairs (SEQ IDs #63 and 64) to create PCR product III (SEQ IDs #65 and #66). For multiplexing standard Illumina index primers #67 and #68) are introduced to yield PCR product IV (SEQ IDs #71 and #72) according to standard Illumina NGS sample preparation protocol.

Results:

Fig S13: Results of NGS sequencing of the model library before and after selection Burrows-Wheeler Alignment and Mapping; NGS platform is an Illumina MiSeq instrument. Data is shown in logarithmic scale, normalized). According to the counts SEQ ID # 8 with a T6 spacer between the binding motifs is most abundant, whilst a SEQ ID #9 with a T12 spacer is slightly diminished. If no spacer is used the enrichment in the final pool is negligible (SEQ ID #7) probably due to steric hinderance. Especially a modification in the G15D motif reduces the abundance in the final pool after selection (SEQ ID # 9 versus #10). Modification in the other binding motif TBA27 also reduces the counts (SEQ ID #11,12 and #13), but with a less significant impact. Modifications in both binding motifs lead to a significant reduced number of reads (SEQ ID #14).

5. Disassociation rates by surface plasmon resonance

Disassociation rates were determined using a G.E. Healthcare Biacore X100. Biotinylated human alpha-thrombin (2 µg/µl, Thermo Fisher Scientific cat. no. RP43103) was diluted with running buffer (10 mM HEPES-KOH, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 0.01% Tween) to ~1µg/ml and immobilized on the active lane of the Sensor Chip SA (G.E. Helathcare cat. no. BR100032) using the in-built wizard application. Two separate chips were immobilized with different levels of thrombin (615.5 and 2452.4 RU; ‘low’ and ‘high’ respectively) and the reference lane was left blank. SEQ ID 15, 16, 20, 21 and 22 were freezed dried and resuspended in running buffer before serial two-fold dilution (max-min concentration range = 1000 to 7.8 nM for ‘low’ immobilization chips and 125 to 7.8 nM for ‘high’ immobilization chips). Samples were injected over both surfaces at a flow rate of 30 µl/min for 180 s before disassociation was monitored for 600 s. For all sequences, expect 15, the surface was regenerated using a 100 mM sodium acetate (pH 4.4) solution containing 4 M MgCl2 at a flow rate of 30 µl/min for 30 s (high immobilization surface) or 15 s (low immobilization surface) before post-regeneration running buffer equilibration time of 180 s.

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For data analysis, the active lane signal was reference subtracted and blank injection subtracted. The decay component for each aptamer was then normalized to the 180 s RU (start of decay) and averaged over the various concentration injections (error in the decay was less than 0.04 of the

normalized response). Decays were fitted to the equation where 𝑅𝑒𝑠𝑝𝑜𝑛𝑐𝑒 = 𝐴 ((1 ‒∝ )𝑒‒ 𝑡𝑘𝑑,1 + ∝ 𝑒

‒ 𝑡𝑘𝑑,2)the fractional contribution , A is the pre-exponential constant, t is time and is the disassociation 𝛼 < 1 𝑘𝑑rate. The goodness of fit was evaluated by considering the weighted residual deviation from the model. Least squared minimization was performed using trust-region-reflective algorithm and custom written code in MatLab 2016a. The decay curves from the high and low immobilization chips were analysed separately.

Fig S14: Average normalised decay curves for monomeric (ID 15, 16) and bivalent aptamers (ID 20-22) from surface plasmon resonance using a high immobilisation surface. The aptamers and raw data are colour-coded, with the modelled fit to a bi-exponential decay in black. The fractional contribution of each disassociation component is in brackets. Immobilized biotinylated human alpha-thrombin = 2452.4 RU

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Mass spectrometry and UV analysis of modified oligonucleotides used in this study:

Mass spectrum and UV analysis of oligonucleotide 1 in table S1. Calculated; 19212, found; 19212.

25


26


27


28

Mass spectrum and UV analysis of oligonucleotide 5 in table S1. Calculated; 18930, found;18929.

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References

[1] T. R. Chan, R. Hilgraf, K. B. Sharpless, V. V. Fokin, Org. Lett. 2004, 6, 2853-2855.[2] A. H. El-Sagheer, A. P. Sanzone, R. Gao, A. Tavassoli, T. Brown, Proc. Natl. Acad. Sci. U. S. A.

2011, 108, 11338–11343.[3] R. D. Langdell, R. H. Wagner, K. M. Brinkhous, J. Lab. Clin. Med. 1953, 41, 637-647.[4] A. J. Quick, M. Stanley-Brown, F. W. A. Bancroft, Am. J. Med. Sci. 1935, 190.

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